This invention relates to the production of adipic acid and precursors thereof by the conversion of biomass-derived carbon sources. More particularly this invention is directed to the biocatalytic conversion of glucose and other sugars capable of being used in the biosynthesis of aromatic amino acids to adipic acid via 3-dehydroshikimate dehydratase, protocatechuate decarboxylase, and catechol 1, 2 dioxygenase, followed by hydrogenation.
Annual world-wide production of adipic acid in 1989 was estimated at 4.2 billion pounds. With U.S. production at 1.75 billion pounds in 1992, adipic acid consistently ranks as one of the top fifty chemicals produced domestically. Nearly 90% of domestic adipic acid is used to produce nylon-6,6. Other uses of adipic acid include production of lubricants and plasticizers, and as a food acidulant.
The dominant industrial process for synthesizing adipic acid employs initial air oxidation of cyclohexane to yield a mixture of cyclohexanone (ketone) and cyclohexanol (alcohol), which is designated KA. Hydrogenation of phenol to yield KA is also used commercially, although this process accounts for just 2% of all adipic acid production. KA produced via both methods is oxidized with nitric acid to produce adipic acid. Reduced nitrogen oxides including NO.sub.2, NO, and N.sub.2 O are produced as by-products and are recycled back to nitric acid at varying levels.
These processes are not entirely desirable due to their heavy reliance upon environmentally sensitive feedstocks, and their propensity to yield undesirable by-products. Cyclohexane is derived from benzene, a known carcinogen which is obtained from nonrenewable fossil fuels. Cyclohexane itself is currently under investigation as a toxic material. Moreover, nitric acid oxidation has been reported to account for 10% of the global increase in atmospheric nitrous oxide. Nitrous oxide has been implicated in the depletion of the ozone layer.
Extensive research has been directed at alternative processes of adipic acid synthesis, though none have been commercialized. Reactions involving cobalt-catalyzed air oxidation of cyclohexane directly to adipic acid or oxidation of cyclohexane with ozone have been examined as methods which avoid intermediate production of KA. Here again, the use of cyclohexane and phenol as feedstocks is undesirable from an environmental standpoint.
Research has also focused on synthesis of adipic acid from alternative feedstocks. Significant attention has been directed at carbonylation of butadiene. More recently, a method of dimerizing methyl acrylates was reported, opening up the possibility of adipic acid synthesis from C-3 feedstocks.
Exploitation of biological systems has also been examined. A strain of Pseudomonas putida has been developed which catalyzes conversion of toluene to cis, cis-muconic acid, which can be hydrogenated to afford adipic acid. However, this method is similar to traditional chemical technology in that it begins with toluene, an environmentally undesirable feedstock. Strains of Acinetobacter and Norcardia have been reported which, when grown on cyclohexanol as the sole source of carbon, produce adipic acid as an intermediate in the metabolic pathway.
Alternatively, strains of Norcardia and Pichia carboniferus have been reported which synthesize adipic acid from diaminododecane and myristic acid, respectively. However, processes based upon these strains are commercially unattractive because starting materials are particularly expensive. Moreover, the biochemical reactions and the induction of enzymatic activities have not been clearly elaborated.
One process combining elements of biocatalysis and chemistry entails the multi-step chemical conversion of biomass into 1,6-hexanediol, which is then oxidized to adipic acid by Gluconobacter oxydans. While this process relies on inexpensive starting materials, it requires multiple chemical conversions which are carried out at elevated temperatures (100.degree. C.-350.degree. C.) and pressures (up to 20,000 psi) and employs multiple metal catalysts including copper chromite.
It would be desirable to provide a synthesis route for adipic acid which not only avoids reliance on environmentally sensitive starting materials but also makes efficient use of inexpensive, renewable resources. It would further be desirable to provide a synthesis route for adipic acid which avoids the need for significant energy inputs and which minimizes the formation of toxic by-products.
The present invention provides methods for the microbial biosynthesis of adipic acid from readily available carbon sources capable of biocatalytic conversion to erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP) in microorganisms having a common pathway of aromatic amino acid biosynthesis. One preferred carbon source is D-glucose. Advantageously, D-glucose, and other carbon sources useable in connection with the present invention, are non-toxic. Furthermore, they are renewable resources derived from starch, cellulose, and sugars found in corn, sugar cane, sugar beets, wood pulp, and other biomass resources.
Host microbial organisms suitable for carrying out the present invention belong to genera possessing an endogenous common pathway of aromatic amino acid biosynthesis. Preferred host organisms are mutant strains of Escherichia coli genetically engineered to express selected genes endogenous to Klebsiella pneumoniae and Acinetobacter calcoaceticus. One preferred E. coli mutant for use in this invention is E. coli AB2834, an auxotrophic mutant which is unable to catalyze the conversion of 3-dehydroshikimate (DHS), an intermediate along the common pathway, into shikimic acid and thereafter into chorismate due to a mutation in the aroE locus which encodes shikimate dehydrogenase.
The common pathway of aromatic amino acid biosynthesis produces the aromatic amino acids, phenylalanine, tyrosine, and tryptophan in bacteria and plants. The common pathway ends in the branch point molecule chorismate, which is subsequently converted into phenylalanine, tyrosine, and tryptophan by three separate terminal pathways.
Approaches for increasing the efficiency of production of the common pathway have been described in U.S. Pat. No. 5,168,056 (issued Dec. 1, 1992) and in U.S. patent application Ser. No. 07/994,194, filed Dec. 21, 1992, the disclosures of which are hereby expressly incorporated by reference.
In using the genetically engineered, mutant host organisms to produce adipic acid according to this invention, carbon flow directed into aromatic amino acid biosynthesis proceeds along the common pathway to yield elevated intracellular levels of the DHS intermediate, which accumulate due to a mutation along the common pathway of aromatic amino acid biosynthesis which prevents the conversion of DHS to chorismate. The DHS intermediate serves as a substrate for the enzyme 3-dehydroshikimate dehydratase to produce protocatechuate. Protocatechuate is thereafter converted to catechol with protocatechuate decarboxylase. Catechol is in turn converted to cis, cis-muconic acid by the action of catechol 1, 2-dioxygenase. Synthesized cis, cis-muconic acid accumulates extracellularly and can be separated from the cells by centrifugation. Cis, cis-muconic acid is thereafter directly hydrogenated to yield adipic acid.
Preferably, the enzymes catalyzing the biosynthesis of cis, cis-muconic acid are expressed in the host cell with recombinant DNA comprising genes encoding the enzymes under control of a constitutive promoter. Carbon flow is thereby forced away from the common pathway, into the divergent pathway to produce cis, cis-muconic acid.
In contrast to known multi-step, energy-intensive conversion processes, the processes of this invention rely on a single-step microbial conversion at relatively low temperature (e.g. about 37.degree. C.) and atmospheric pressure, followed by a single chemical transformation performed at ambient temperatures at mild pressures (50 psi) with a platinum catalyst. Moreover, a 90% or higher conversion of the biosynthesized cis, cis-muconic acid to adipic acid can be achieved.
In one preferred embodiment using the host strain E. coli AB2834, DHS intracellular concentrations are increased due to a mutation in a gene (aroE) which encodes shikimate dehydrogenase. DHS is transformed to catechol along a divergent pathway enabled by transformation of the host cell with expressible genetic fragments encoding DHS dehydratase and protocatechuate decarboxylase and with genes encoding for enzymes which commit an increased amount of carbon to the common pathway of aromatic amino acid biosynthesis. Further transformation of the host cell with expressible genetic fragments encoding catechol 1, 2-dioxygenase enables the biocatalytic conversion of catechol to cis, cis-muconic acid, which, upon separation from the cells, is directly hydrogenated over 10% platinum on carbon at 50 psi hydrogen pressure for three hours at room temperature to generate adipic acid. Analysis of culture supernatants of recombinant mutants of this invention using nuclear magnetic resonance spectroscopy (NMR) demonstrates that adipic acid is the primary product. A 90% conversion of cis, cis-muconate into adipic acid can be achieved.
Additional objects, features, and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.